Multi-analyte reference solutions with stable pO2 in zero headspace containers

ABSTRACT

Multi-analyte reference solutions having a stable partial pressure of oxygen (pO2) in zero headspace packaging and methods for preparing such solutions are disclosed. The solutions have long shelf and use lives when stored at room temperature and are packaged in laminated foil containers having low or no oxygen reactivity. Access devices are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/641,033, filedAug. 16, 2000 now U.S. Pat. No. 6,632,675 issued Oct. 14, 2003, which isa divisional of U.S. patent application Ser. No. 09/018,137, filed Feb.3, 1998, now U.S. Pat. No. 6,136,607 issued Oct. 24, 2000, which is acontinuation-in-part of U.S. patent Ser. No. 08/740,410, filed Oct. 29,1996, now U.S. Pat. No. 5,780,302 issued Jul. 14, 1998 and whichdeclares priority from U.S. Provisional Application No. 60/006,742,filed Nov. 2, 1995, which is now abandoned, the disclosures of which arehereby incorporated by reference. Priority from the above U.S. patentand applications is claimed for this application under 35 U.S.C. §120.

TECHNICAL FIELD

This invention relates primarily to the field of clinical referencesolutions—quality control reagents and calibrators. More specifically itrelates to methods of preparing multi-analyte reference solutions thathave stable oxygen partial pressure (pO₂) in zero headspace containers,preferably in flexible foil laminate containers. The solutions arestable at room temperature and have long shelf and use lives.

BACKGROUND

Clinical laboratories employ a variety of instrument systems for theanalysis of patient samples. For example, pH/blood gas instrumentsmeasure blood pH, pCO₂ and pO₂. CO-Oximeter instruments typicallymeasure the total hemoglobin concentration (tHb), and the hemoglobinfractions—oxyhemoglobin (O₂ Hb), carboxyhemoglobin (COHb), methemoglobin(MetHb), reduced hemoglobin (HHb) and sulfhemoglobin (SHb)(collectivelyreferred to as “CO-Ox fractions”). Ion selective electrode (ISE)instruments measure the content of blood electrolytes, such as, Na⁺,Cl⁻, Ca⁺⁺, K⁺, Mg⁺⁺ and Li⁺. Also, a variety of other parameters suchas, metabolites, e.g., glucose, lactate, creatinine and urea, can bemeasured in clinical laboratories by related instrument systems.

Instrument systems currently available may combine the measurement ofblood pH, gases, electrolytes, various metabolites, and CO-Ox fractionsin one instrument for a comprehensive testing of the properties ofblood. For example, all such analytes are measured by the Rapidlab™ 865critical care diagnostics system from Chiron Diagnostics Corporation[Medfield, Mass. (USA)].

A calibrator is used to set the response level of the sensors. A controlis used to verify the accuracy and reliability of such aninstrumentation system.

A control is a solution having a known concentration of an analyte oranalytes contained in the same, or a similar matrix in which the samplesto be analyzed exist. The assay results from the control product arecompared to the expected assay results to assure that the assaytechnique is performing as expected.

Commercial blood gas analysis systems have been available since the1960s. The earliest reference materials were gas mixtures in pressurizedcylinders, and those materials are still commonly used. In the 1970s,the development of liquid reference solutions began, leading to productsin which reagents have been equilibrated with precision gas mixtures andpackaged in flexible containers with zero headspace, requiring eitherrefrigeration to maintain stability or the resort to calculations tocompensate for the expected pO₂ changes during storage.

Most quality control materials for such analyzers consist of tonometeredaqueous solutions (a solution containing dissolved gases) in glassampules. The typical gas headspace above the liquid in those ampulesprovides a reserve of oxygen against any potential oxygen-consumingreactions which may occur within the solution during the shelf life ofthe product.

In the absence of a gas headspace within their containers, referencesolutions for oxygen determinations are particularly difficult to makeand maintain stable. The inventors determined that the sources of saidinstability could be several.

First, the instability may be due to reactivity between the dissolvedoxygen and the other components of the calibrator or quality controlreagent. The other components might either react with the dissolvedoxygen, reducing its concentration, or, alternatively, the othercomponents may react with each other to generate oxygen, thus alsochanging the oxygen concentration. Second, the solution might becontaminated with microorganisms which, due to their metabolism, mightchange the oxygen content. Third, the oxygen might permeate through, orreact with, the packaging material, also affecting the oxygen content ofthe reference material.

Reference materials that are manufactured for distribution in commercemust be made to withstand the various conditions encountered in thedistribution chain and must be sufficiently stable to provide goodperformance within the time frame in which they are expected to be usedby the customer, which is usually at least about six months, preferablyfor about nine months, and more preferably approximately 1 year for thetypical calibrating or quality control solution distributed tocommercial laboratories and hospitals. In addition, reference solutions,as with other reagents, should be packaged in containers which are easyto handle, convenient to use and which meet other design requirements oftheir intended usage. This is particularly true of reagents which areused in conjunction with various analytical instruments.

The users of instruments which determine the oxygen partial pressure ofblood and other body fluids have a need for such reference materials andwould benefit from liquid materials over the more conventional precisiongas mixtures in cylinders with regulators. Liquid reference solutionsare inherently less expensive, safer, and easier to manipulate thanhigh-pressure gas tanks.

Although reference solutions used in instruments measuring pO₂ have beenmade in the past, they have suffered from being unstable and havingexpensive, complicated, or unreliable means to access their contents.Some reference solutions, when used on analytical instruments, haveextended their usefulness by allowing the instrument to calculate theexpected oxygen level, said level being calculable from the age of theproduct, given the fact that the rate of decrease in oxygen level can bepredicted based on historic performance [Conlon et al., Clin. Chem., 42:6—Abstract S281 (1996)]. Several developers have included inner layersof plastic materials selected because of their heat sealability (e.g.,U.S. Pat. No. 5,405,510—Betts) or low gas permeability (U.S. Pat. No.4,116,336—Sorensen) or gas tightness (U.S. Pat. No. 4,163,734—Sorensen).Some have disclosed that the inner layer should be inert, but have notprovided enablement as to how to select such an inner layer (U.S. Pat.No. 4,643,976—Hoskins) and/or weren't capable of maintaining oxygen at aprecise level appropriate for blood gas purposes.

Most blood gas/electrolyte/metabolite/CO-Oximetry/hematocrit qualitycontrols (QCs) on the market today are provided in glass ampules whichmust be manually broken and manually presented to the analyzer. Ruther,H., U.S. Pat. No. 5,628,353 (issued May 13, 1997) describes an automateddevice which breaks open glass ampules by forcing a metal tube withthick walls and a small inner diameter, into the bottom of an ampule,and then aspirates the contents of the ampule into an analyzer. Such anautomated ampule breaker is mechanically complex, requiring moving partsthat are subject to wear and risk of failure, and could be subject tojamming and clogging from small bits of broken ampule glass.

In the 1980s, Kevin J. Sullivan disclosed an alternative to glassampules—the first commercial product with a blood gas reagent in aflexible, zero headspace package [U.S. Pat. Nos. 4,266,941; 4,375,743;and 4,470,520]. Coated aluminum tubes were filled with 40-50 mL of bloodgas QC solutions without any headspace. The tubes were enclosed inpressurized cans, to prevent outgassing and to supply a source of forceto cause the QC solutions to flow into the sample path of a blood gasanalyzer. One container of Sullivan's packaging design replaced about 30glass ampules. Sullivan's packaging relieved the user of the task ofopening many glass ampules and of the attendent risks of broken glass.The disadvantages of Sullivan's packaging included a need torefrigerate, a shelf life of less than a year, a menu of only threeanalytes, and the complexity and cost of a spring-loaded valve.

The instant invention not only overcomes the limitations of glassampules, such as sensitivity of gas values to room temperature due tothe headspace above the liquid, and complications resulting from thesharp edges which form upon breaking them open, or from the small, sharpglass pieces which can break off during ampule opening, but alsoovercomes the limitations of Sullivan's zero headspace packagingdescribed above. The multi-analyte reference solutions with stable pO₂of the instant invention are packaged in containers with zero headspace,preferably in flexible foil laminate containers, and are stable at roomtemperature for a shelf life of from about one to three years.

An additional shortcoming of storage devices for reference solutions foroxygen determinations (oxygen reference solutions) has been the openingor valve required to access the fluid for use, while maintaining theintegrity of the fluid during storage. The materials available for valveconstruction and the need to breach the barrier layer to incorporate thevalve may have compromised fluid stability. The access device disclosedherein for the preferred foil laminate containers used in the methods ofthe invention solves that problem. The simplicity of the one-piece valveshould result in cost savings and greater reliability.

Further the multi-analyte reference solutions with stable pO₂ in zeroheadspace containers of this invention provide cost savings in that onesuch container can be the equivalent of a box of 30 or more ampules thatare currently on the market. Further cost savings are provided in theconsolidation of formulations in 5 level quality control (QC) reagentsof this invention which are useful to control from about 5 to about 20analytes. Providing a reduced number of formulations to control forpH/blood gas/electrolyte/metabolite/total hemoglobin (tHb)/hematocritand CO-Oximetry analytes saves time on an analyzer system, allowing formore patient samples to be assayed, and consequently minimizes assaycosts.

SUMMARY OF THE INVENTION

One object of this invention was to overcome the shortcomings of glassampules as storage containers for QCs and calibrators used with wholeblood analyzers, while allowing for automation of QC and calibratordelivery. In one aspect, the instant invention overcomes problemspresented by glass ampules as storage containers for oxygen referencesolutions used as controls for instruments that measure blood analytes.Disclosed herein is a novel flexible package for oxygen referencesolutions.

The package is made from a laminated film comprising an inner layer withlow or no oxygen reactivity, preferably polypropylene, aluminum foil asthe middle layer, and an outer layer that protects the aluminum foillayer from physical damage, e.g., abrasion or corrosion. The seams areheat sealed, while an optional access device for allowing access to thesolution after the storage period, is attached to the inside wall of thebag without breaching the laminated layers. The foil laminate packagingallows for mechanical simplicity.

Preferred tubing for conveying a multi-analyte reference solution withstable pO₂ from a container to a blood analyzer is also disclosed. Suchtubing is flexible and relatively gas impervious, having a durometer(Shore D scale) in the range of 10 to 100, preferably from 70 to 94 andmore preferably from 80 to 84. Preferred for such tubing are polyamidecondensation polymers, more preferred are polyester/polyether blockco-polymers or polyester elastomers, and especially preferred are Nylon™[DuPont; Wilmington, Del. (USA)] and Hytrel™ 8238 [DuPont].

Another object of this invention is to provide multi-analyte referencesolutions with stable pO₂ in zero headspace containers, wherein thesolutions are stable at room temperature for at least six months,preferably for at least nine months, more preferably for at least abouta year, still more preferably for more than a year, and even morepreferably for from two to up to three years. The most unstablecomponent of a multi-analyte reference solution in a zero headspaceenvironment used for oxygen determinations, among other analyses, isusually the pO₂. Methods are provided to maintain the pO₂ of such amulti-analyte reference solution within a predetermined range. Centralto those methods is the principle of minimizing contact of the oxygen inthe reference solution with materials that are oxygen reactive.

The lining of the preferred foil laminate packaging of this inventionthat contains the multi-analyte reference solutions with stable pO₂ ofthis invention is selected for its low reactivity to oxygen. Thepreferred polypropylene lining of the foil laminate package, preferablya foil laminate pouch, was chosen as it is essentially inert to oxygen.

Further, source materials, particularly organic source materials, forthe other components of the multi-analyte reference solutions withstable pO₂ of this invention are also screened for low oxygenreactivity. It was found that some source materials contain impuritiesthat are oxygen reactive enough to destabilize the pO₂ of suchmulti-analyte reference solutions.

It is further an object of this invention to prepare a panel ofmulti-analyte reference solutions with stable pO₂ that control fromabout 5 to about 20 analytes in as few containers as practicable, forexample, a quality control reagent in five foil laminate containers (a 5level QC reagent), wherein there is a different formulation in each zeroheadspace container. Key to combining so many critical analytes in asfew containers as practicable are (1) using a low pH/low pO₂/lowglucose/low tHb formulation as an all-inclusive level; and (2)separating the mid-pO₂ and high-pO₂ reference solutions from glucose andfrom the dyes needed to simulate tHb and/or CO-Ox fractions.

A pH range considered low for the multi-analyte reference solutions ofthis invention is from about 6.4 to about 7.4. Exemplary of a low pO₂range is from about 20 mmHg to about 75 mmHg. Exemplary of a mid-pO₂ tohigh pO₂ range is from about 80 mmHg to about 600 mmHg. An exemplary lowglucose concentration is from about 10 mg/dL to about 80 mg/dL. Anexemplary low dye concentration corresponds to a tub concentration offrom about 5 g/dL to about 11 g/dL.

Methods of preparing such reagents are disclosed as well as the reagentsprepared by those methods. Further disclosed are representativeembodiments of such a quality control reagent constituting fiveformulations (a 5 level QC reagent).

Although exemplified herein are uses for the multi-analyte referencesolutions laminate with stable pO₂ in zero headspace containers of thisinvention in the clinical field, they may also be used in theenvironmental and biotechnological fields, among other fields thatrequire oxygen analysis. For example, the solutions of this inventionwould be useful in fermentation analyses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a side view of a four-sided multilayer package of thisinvention. FIG. 1b is a cross-sectional view showing three layers of thepackaging. FIG. 1c is a first end view of the package of FIG. 1a. FIG.1d is a frontal view of a three-sided, center seam package.

FIG. 2 is a side view of an access device used in the methods of thisinvention.

FIG. 3 is a side view of a probe which pierces the foil laminate andfits into the access device of FIG. 2.

FIG. 4a is a diagram of a clamp and locating device that can be used inconjunction with the foil laminate containers of this invention. FIG. 4bis a top view of the device of FIG. 4a. FIG. 4c is a side view of thedevice of FIG. 4a.

FIG. 5 is an Arrhenius diagram showing the predicted shelf life of atypical formulation contained in the novel packaging of this invention.

FIG. 6 graphically demonstrates a use life study wherein pO₂ of arepresentative automated quality control formulation over time wasmeasured, wherein the tubing used to convey solutions from the pierceprobe to the fluidic selection valve of the foil laminate pouch waseither Nylon™ [DuPont; Wilmington, Del., USA]or Hytrel™ 6356 [Dupont].

ABBREVIATIONS AND BRAND NAMES

AQC automated quality control reagent Brij 700 ™ polyoxyethylene 100stearyl ether with 0.01% BHA and 0.005% citric acid as preservatives,[surfactant from ICI Americas, Inc., Wilmington, Del., USA] CDC ChironDiagnostics Corporation (formerly Ciba Corning Diagnostics Corporation)COHb carboxyhemoglobin CO-Ox CO-Oximeter or CO-Oximetry for instrumentand method, respectively of measuring total hemo- globin and hemoglobinfractions, such as, O₂ Hb, MetHb, COHb, SHb and HHb Cosmocil CQ ™polyhexamethylene biguanide hydrochloride, 20% [biocide from ZenecaBiocides, Wilmington, Del. (USA)] Dantogard ™ 32%1,3-bis(hydroxymethyl)-5,5-dimethylhydan- toin and 7.5%hydroxymethyl-5,5-dimethylhydantoin, in water [biocide from Lonza, Inc.,Fair Lawn, N.J., (USA)] EDTA ethylene diamine tetraacetate Hcthematocrit HDPE high-density polyethylene HEPES2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid [pKa of 7.31 at37° C.] HHb reduced hemoglobin HIDA N-(2-hydroxyethyl)iminodiacetic acidISE ion-selective-electrode LLDPE linear low-density polyethylence M288Model 288 Blood Gas Analyzer [Chiron Diagnostics Corporation; Medfield,Mass. (USA)] MetHb methemoglobin MIT methylisothiazolone [a biocide fromBoehringer- Mannheim GmBH, Indianapolis, Ind. (USA)] MOPS3-(N-morpholino)propanesulfonic acid [pKa of 7.01 at 37° C.] M. Yellow 7Mordant Yellow 7 O₂ Hb oxyhemoglobin P.B. Violet Patent Blue Violet PEpolyethylene pCO₂ partial pressure of carbon dioxide pO₂ partialpressure of oxygen PP polypropylene ProClin 300 ™ 2.3% of5-chloro-2-methyl-4-isothiazolin-3-one and 0.7% of2-methyl-4-isothiazolin-3-one with 3% alkyl carboxylate in 94% of amodified glycol [biocide from Rhom & Haas Co., Spring House, Pa. (USA)]PSI pounds per square inch PVC polyvinylchloride PVF polyvinylfluorideQC quality control Saran ™ polyvinylidene chloride [Dow ChemicalCompany, Midland, Mich. (USA)] SHb sulfhemoglobin SRB sulforhodamine B(dye; CAS #3520-42-1) THb total hemoglobin TTF time to failure

DETAILED DESCRIPTION OF THE INVENTION Foil Laminate Packaging

In one aspect, this invention concerns novel flexible packaging foroxygen reference solutions. Typical oxygen reference solutions used inwhole blood analyzers comprise sodium, potassium, and calcium chloridesalts, pH buffer, sodium bicarbonate, calcium chelating agent,surfactant, and biocide, which are equilibrated under partial vacuumwith a carbon dioxide/oxygen gas mixture before filling. The typicaloxygen partial pressures are from about 30 up to about 700 mmHg, butpartial pressures as high as 2000 mmHg (i.e., greater than ambient) canbe used, as well as partial pressures as low as zero (no oxygenpresent).

The packaging described herein stabilizes the oxygen reference solutionsvia the use of a multilayered film as the packaging material. Inaddition, the package incorporates an unusual access device for removingthe solution. The access device is not exposed to the outside of thecontainer. Instead it is sealed within the container and, as a result,does not provide an opportunity for there to be leakage around the sealduring the pre-use storage as opposed to having the access device sealedwithin the package seam or through the wall of the container, where onewould ordinarily expect it to be sealed.

The foil laminate packaging described herein is novel. First, thepackaging material is selected because of the non-reactivity of itsinner layer with oxygen. Second, the thickness of its layers aredifferent from those of previous flexible packages. Third, the packagedescribed herein has an optional, novel valve or access device, whichreduces the amount of leakage and better maintains the integrity of thecontents of the container. Fourth, all prior art in this area oftechnology was based on 4-sided bags with the security of one continuousseal around the entire perimeter of the package; whereas disclosedherein is a 3-sided, center-seal pouch having in places two, in otherplaces four, layers of laminate to seal through, and six stress pointsper bag where laminate is folded at 360° and where one might thereforeexpect that a thin channel allowing gas exchange might result.

The foil laminate packaging of this invention is filled under vacuumwithout any headspace of gas above the oxygen reference liquid in orderto make the contents insensitive to temperature and barometric pressurechanges. A suitable fill volume would be between 10 and 1000 mL, andpreferably about 20 to 250 mL.

Below under the heading Film, the multilayered foil laminate packagingis described in detail. The access device is similarly described indetail below under the heading The Access Device.

Multi-Analyte Reference Solutions with Stable pO₂

In another aspect, this invention concerns methods of preparingmulti-analyte reference solutions with stable pO₂ in zero headspacecontainers, preferably in the flexible foil laminate packaging describedherein. The phrase “multi-analyte reference solution with stable pO₂” isherein defined to mean a reference solution used as a calibrator or as acontrol for pO₂ plus one or more other analytes, wherein the pO₂ of saidreference solution is maintained within a predetermined range. Exemplaryof such a range is at a specified value ±4 mmHg, alternatively at aspecified value ±2%, preferably ±1%.

Examples of multi-analyte reference solutions with stable pO₂ includethe following: (1) a blood gas reference solution with a stable pO₂which calibrates or controls for pO₂, pH and pCO₂; (2) a blood gas andelectrolyte reference solution which calibrates or controls for pO₂, pH,pCO₂ and electrolytes, such as, Na⁺, Cl⁻, K⁺, Ca⁺⁺, Li⁺ and Mg⁺⁺; (3) ablood gas/electrolyte and metabolite reference solution which calibratesor controls for pO₂, pH, pCO₂, electrolytes, and metabolites, such as,glucose, lactate, bilirubin, urea and creatinine; (4) a bloodgas/electrolyte/metabolite and tHb reference solution; (5) a bloodgas/electrolyte/metabolite/tHb and CO-Ox fraction reference solution;(6) reference solutions used for oxygen determination and to control orcalibrate for one or more other analyte(s) selected from pH, CO₂,electrolytes, metabolites, tHb, CO-Ox fractions, and Hct.

Exemplary of pO₂ ranges calibrated or controlled by the multi-analytereference solutions with stable pO₂ of this invention are those between0 to 1000 mmHg, 20 to 700 mmHg and 30 to 500 mmHg. Exemplary pCO₂ rangescalibrated or controlled by the multi-analyte reference solutions ofthis invention that test for blood gas are those between 0 to 150 mmHg,5 to 100 mmHg and 15 to 75 mmHg.

Described below under the heading Methods of Preparing Multi-AnalyteReference Solutions with Stable pO₂ are methods for maintaining the pO₂of an multi-analyte reference solutions with stable pO₂ within apredetermined range for a desirable shelf life of from one to aboutthree years.

Described below under the sub-heading Analyte Levels and Formulations ofRepresentative OC and Calibrator Reagents, are exemplary and preferredfive level QC reagents of this invention. Parameters of a keyall-inclusive level (exemplified by level 3 below) are set forth underthat sub-heading.

Methods of Preparing Multi-Analyte Reference Solutions with Stable pO₂

The most unstable component of a multi-analyte clinical referencesolution in a zero headspace container used for oxygen determinations,among other determination(s), is usually pO₂. Methods are provided tomaintain the pO₂ of multi-analyte reference solutions in a zeroheadspace container within a predetermined range, that is, e.g., at aspecified value ±4 mmHg, alternatively ±2%, preferably at ±1%.

Central to the methods of maintaining the stability of pO₂ inmulti-analyte reference solutions in zero headspace containers isminimizing the contact of the oxygen in such a reference solution withmaterials that are oxygen reactive. As detailed infra, the lining of thefoil laminate packaging for multi-analyte reference solutions withstable pO₂ of this invention is selected for its low reactivity tooxygen. PP is the preferred lining material for the flexible zeroheadspace packaging of this invention.

Further the methods of this invention for preparing multi-analytereference solutions with stable pO₂ comprise preparing such referencesolution formulations with components that have been screened for low orno oxygen reactivity. A representative raw material screening process isprovided below. Particularly important is the screening of organicmaterials for low or no oxygen reactivity. It was found, as shown below,that some source materials may contain impurities that are oxygenreactive enough to destabilize the pO₂ of such a multi-analyte referencesolution in a zero headspace container.

Further are provided methods of preparing multi-analyte referencesolutions with stable pO₂ in the least number of zero headspacecontainers for detecting as many critical care analytes as practicable.Set forth below are examples of such formulations. Again low oxygenreactivity is critical to preparing stable formulations. It is importantto formulate an all-inclusive level, wherein the pO₂ is low, forexample, at 30 mmHg, 40 mmHg or at 50 mmHg, at a low pH, for example, atpH 7.13 or 7.15, and at a low glucose concentration, for example, at 46or 50 mg/dL, and at a low dye concentration.

Further in regard to other levels of such a reagent, it is important toseparate the formulations used to test for mid-pO₂ and high-pO₂ fromglucose and from the dyes needed to simulate tHb and CO-Ox fractions.Exemplary formulations are provided below.

Analyte Levels and Formulation of Representative OC and CalibratorReagents

It is desirable to prepare a minimum number of formulations for themulti-analyte reference solution panels of this invention, [i.e.,preferred quality control (QC) reagents] so that, test time on analyzersis maximized and costs are minimized. However, the lack of headspace inthe packaging of this invention renders that goal of minimizing thenumber of formulations to test a maximum number of analytes difficult inthat unlike the conventional glass ampule packaging which has on avolume-to-volume basis, roughly 32 times more oxygen in the headspacethan in solution, the packaging of the instant invention has no oxygenreserve. Without an oxygen reserve, organic materials in the solutions,such as, glucose and the dyes used to simulate hemoglobin, or impuritiesin such source materials, react with the oxygen present in thesolutions, thereby reducing the pO₂ of the solutions.

Key to combining so many critical analytes in as few containers aspracticable are (1) using a low pH/low pO₂/low glucose/low tHbformulation as an all-inclusive level (exemplified by level 3 herein);and (2) separating the mid-pO₂ and high-pO₂ reference solutions fromglucose and from dyes. Exemplary formulations for a five level qualitycontrol reagent are provided below. Such a five level QC combines fromabout 5 to about 20 analytes, preferably from about 12 to about 20analytes including pH, pO₂, pCO₂, electrolytes, metabolites, hematocrit,tHb, and CO-Ox fractions. The all-inclusive level of such a QC reagentcontrols for the following analyte levels:

(1) a low pH, from about 6.4 to about 7.4, more preferably from about6.8 to about 7.3, still more preferably from about 7.1 to about 7.2;

(2) a pO₂ of from about 20 mmHg to about 75 mmHg, more preferably fromabout 25 mmHg to about 70 mmHg, and still more preferably from about 30mmHg to about 60 mmHg; and

(3) a low glucose concentration of from about 10 mg/dL to about 80mg/dL, more preferably from about 30 mg/dL to about 60 mg/dL; and

(4) contains a low dye concentration corresponding to a hemoglobinconcentration of about 5 g/dL to about 11 g/dL, preferably from about 6g/dL to about 10 g/dL, more preferably from about 7 g/dL to about 9g/dL.

Table 1 below shows exemplary analyte levels for a representative 5level automatic quality control reagent (“5 Level AQC”) of thisinvention.

TABLE 1 Exemplary Analyte Levels for 5-level AQC Analyte 1 2 3 4 5 pH7.55 7.35 7.05 pCO₂, mmHg 20 40 70 pO₂ 150 100 50 Na⁺, mmol/L 155 135115 K⁺, mmol/L 7.0 5.0 3.0 Ca⁺⁺, mmol/L 0.8 1.2 1.6 Mg⁺⁺, mmol/L 0.4 0.61.0 Cl⁻, mmol/L 120 100 80 Lactate, mmol/L 3 1 12 Glucose, mg/dL 50 100200 Urea, mg/dL 12 70 Creatinine, mg/dL 1.0 7.0 Bilirubin, mg/dL 3 15 25tHb, g/dL 8 14 18 O₂Hb, % 60 92 80 COHb, % 18 3 3 MetHb, % 6 2 14 HHB, %16 3 3 Hct, % 45 25

It is further preferred that analyte levels of the reference solutionsinclude not only tHb as an analyte, but also the other CO-Oxfractions—O₂ Hb, COHb, MetHb SHb and HHb as shown in Table 1. Therefore,16 analytes are controlled by the representative all-inclusive level(Level 3) as follows:

Blood Gas pH, pCO₂, pO₂ Electrolytes Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, Cl MetabolitesGlucose, Lactate, Bilirubin CO-Ox Glucose, Lactate, Bilirubin

Table 2 sets forth representative formulations that could be used toprepare a 5-level AQC. It is preferred that Hct, creatinine and ureaonly be monitored at two levels, whereas the other analytes aremonitored at three levels in five formulations.

TABLE 2 Representative Formulations for 5-Level AQC 1 2 3 4 5 MOPS:mmol/L 30 30 27 10 30 NaOH mmol/L 29 28 27 12 26 NaHCO₃ mmol/L 21 21 216 6 NaCl mmol/L 115 95 75 14 32 KCl mmol/L 7.9 5.7 3.4 4 4 Citric Acidmmol/L 1.5 2.0 2.5 2.0 2.0 CaCl₂ mmol/L 1.8 2.4 3.4 2.4 2.4Mg⁺⁺(Acetate-)₂ mmol/L 0.9 1.2 2.0 1.2 1.2 Li⁺Lactate⁻ mmol/L 3.0 1.012.0 Glucose: g/l 0.50 1.00 2.00 SRB (red dye) g/L 0.490 0.924 1.104 M.Yellow 7 g/L 0.249 1.770 0.786 FD&C Blue #1 g/L .0027 .0259 P.B. Violetg/L 0.103 Creatinine .0100 .0700 Urea g/L 0.257 1.50 Brij 700 ™ g/L .05.05 .05 .05 .05 MIT g/L .40 .40 .40 0.40 0.40 Tonometry gas 6/48 10/2517/5 6/48 6/48 % CO₂/% O₂/Bal. N₂

Exemplary Preferred All-Inclusive Level (Level 3) Formulation

A preferred all-inclusive level (designated Level 3 herein) formulationof a 5-Level AQC would control from about 5 to about 20 analytes,preferably from about 12 to about 18 analytes, more preferably fromabout 14 to about 16 analytes. The following is an exemplary preferredformulation which includes 14 components:

1. MOPS  30 mmol/L 8. Glucose 2.8 mmol/L 2. NaOH  25 mmol/L 9. CitricAcid 2.0 mmol/L 3. NaHCO₃  20 mmol/L 10. SRB  0.49 g/L 4. NaCl  75mmol/L 11. Mordant Yellow 70.25 g/L 5. KCl 3.4 mmol/L 12. FD&C Blue 10.003 g/L 6. CaCl₂ 3.0 mmol/L 13. Brij 700  0.05 g/L 7. Li⁺Lactate 3.0mmol/L 14. ProClin 300  0.5 g/L.

Accelerated stability studies are disclosed below for that preferredall-inclusive (Level 3) formulation.

The following analyte levels were obtained with that preferredall-inclusive (Level 3) formulation:

pH 7.13 Na⁺  120 mmol/L tHb 8.2 g/dl pCO₂ 67 mm K⁺  3.3 mmol/L O₂Hb 14%pO₂ 34 mm Ca⁺⁺ 1.48 mmol/L COHb 70% Glucose 46 mg/dL Cl⁻   87 mmol/LMetHb  1% Lactate 3 mmol/L Hhb 14%.

Bags from throughout the lot were randomly selected and stressed atelevated temperatures for appropriate time intervals in order to performan accelerated stability study and generate an Arrhenius plot to predictshelf life at room temperature. The methods used were similar to thosedescribed infra. Results for pO₂, the least stable analyte, are shown inTable 3.

TABLE 3 Accelerated Stability of an Exemplary Level 3 FormulationTemperature, ° C. Time, wks ΔpO₂ v. control, mmHg 55 1 −4.3 2 −6.4 50 2−1.8 6 −4.0 45 6 −3.3 10 −4.4 Allowable Change ±4

The table below shows the Arrhenius calculations used to derive theestimated

TABLE 4 Arrhenius Calculations for a Preferred Level 3 FormulationTemperature, ° C. 1/K Time-to-Failure, wks Log (ttf) 55 .0030488 1.10.055 50 .0030960 5.8 0.77 45 .0031447 8.5 0.93 25 .0033557 875 2.94

The projected room temperature shelf life of 875 weeks, or 17 years, forthe representative Level 3 formulation was estimated using 0.94 as thecorrelation coefficient. A more conservative estimate can be made usinga rule-of-thumb which relies on the fact that the minimum change inreaction rate per 10° C. increase in reaction temperature is an increaseof two times. Based on failure in 2 months at 45° C., the inventorswould estimate that failure will not occur at 25° C. until at least 8months. However, the inventors consider it highly unlikely that thereaction rate increase per 10° C. increase would be any less than threetimes. Therefore, the inventors consider that a realistic but stillconservative estimate of the shelf life of the representative Level 3formulation would be at least 18 months.

Formulation Preparation

To prepare the formulations of this invention, all solutions requiretonometry with the appropriate gases to achieve the gas levels listedabove. Although gas values are not always listed above for levels 4 and5, tonometry is still desirable in order to achieve gas levels whichminimize hysteresis and drift effects on the gas sensors.

The tonometry can be performed at temperatures such as 25° C. or 37° C.or even 50° C., and of course the choice of temperature will affect thecomposition of the tonometry gas. More importantly, tonometry should beperformed at sub-atmospheric pressures, preferably in the 300-500 mmHgrange, so that outgassing will not occur if the solutions are used athigh altitudes where the barometric pressure is below normal, or in warmenvironments. Obviously, the higher the tonometry temperature, thehigher the pressure allowed in the tonometer. An example of a suitablecondition is 37° C. at 450 mmHg, where the gas composition for a level 2QC would be 10% CO₂, 25% O₂ and 65% N₂.

Exemplary preferred dyes for the formulations of this invention arelisted in Table 2, supra. Those dyes are disclosed in Li, J., EP 0 743523 A2 (published Nov. 20, 1996).

Buffers

HEPES and MOPS are preferred buffers for the formulations of thisinvention. MOPS is a particularly preferred buffer. Other suitablebuffer systems, including the sodium salt derivatives, are described byGood et al., Biochemistry, 5: 467-477 (1966) and Ferguson et al.,Analytical Biochemistry, 104: 300-310 (1980).

Shelf Life and Use Life

One object of this invention is to increase the shelf life and use lifeof the QC and calibrator formulations of this invention. An acceptableshelf life (i.e., closed package) would be about one year. A preferredshelf life would be from about one year to two years, and still morepreferred from about one to three years.

An acceptable use life (i.e., open package) would be about two weeks,preferably from about two weeks to about a month, and more preferablyfrom about two weeks to about two months. The use life is extended byappropriate selection of tubing material to conduct reference solutionsfrom the access device to the blood analyzer as described infra.

The inventors discovered a critical element in how the formulationsimpact shelf life by de-stabilizing pO₂. One study compared a verysimple formulation, containing only sodium bicarbonate to neutralize theCO₂ in the tonometry gas, and Brij 700 surfactant, to create appropriatesurface tension, such that the solution behaves normally in thetonometer and filler, to a complete 10-ingredient formulation. The dataare summarized in Tables 5 and 6.

TABLE 5 Accelerated Stability of 2- vs 10-Ingredient Formulation: ΔpO₂,mmHg from control Only Brij + +8 Other Temp Time, wks Bicarb Chemicals60° C. 1 −5.3 −14.0 55° C. 1 −2.4 −7.7 2 −5.6 −15.4 50° C. 1 −2.6 −3.3 2−2.7 −12.4 45° C. 2 +0.2 −4.7 Allowable Change ±4.4 ±4.4

TABLE 6 Arrhenius Calculation Based on pO₂ Data for Formulations inTable XVI Bicarb + Brij +8 Chemicals Time-to- Time-to- Temp 1/K failureLog (ttf) failure Log (ttf) 60° C. .0030030  5.8 days 0.763  2.2 days0.342 55° C. .0030488  11.3 days 1.054  4.0 days  .0602 50° C. .0030960 22.8 days 1.358  7.1 days 0.854 45° C. .0031447 13.1 days 1.117 25° C..0033557  1042 days 3.018  185 days 2.268

The correlation for the Arrhenius prediction for the 2-componentformulation was 0.99999, and for the 10-component formulation, 0.9999(r). It can be seen that addition of eight additional chemicals, theinorganic compounds NaCl, KCl, CaCl₂, NaOH, and the organic compoundscitric acid, glucose, MOPS (pH buffer), and ProClin 300 (biocide),caused the pO₂ to be less stable by 5-6 times as compared to the simple,2-component formulation. The shelf life estimate for 25° C. decreasedfrom 34 months for the 2-component formulation to 6 months for the10-component formulation. Thus, some or all of the eight added chemicalsreacted with oxygen in the aqueous solution in the flexible bag, causingpremature loss of shelf life.

Therefore, studies by the inventors have shown that it is difficult toachieve stable pO₂ in a zero-headspace package with formulations havingmany ingredients, each potentially capable of reacting with oxygen, andrealizing that interactions among ingredients could also bede-stabilizing. Specifically, test results suggest that glucose and thedyes used to simulate hemoglobin can react with oxygen. The oxygenreactivity of those chemicals is one reason the inventors prefer toseparate those chemicals in QC levels 4 and 5 from QC levels 1 and 2.However, the inventors realize that the QC all-inclusive level (level 3)includes those 3 analytes along with the other nine analytes, butdetermined that that all-inclusive level 3 formulation should workbecause:

1. at pH 7.15, glucose is more stable than at the two higher pH levels;

2. the levels of glucose and Hb-simulating dye are all low; and

3. the pO₂ is low. In fact, the true pO₂ at the low level is roughlyhalf of the measured pO₂.

Thus, the inventors discovered that the unique properties of level 3allow the packaging of a QC in 5 containers rather than 6, provides theadvantage to the customer of more patient samples to be assayed in agiven time period.

Direct Comparison of pO₂ Stability in Zero Headspace Packaging v.Ampules

A study was performed to compare a conventional multi-analyte QCformulation, similar to the formulation in Table A of U.S. Pat. No.5,637,505, in glass ampules to that same formulation in a zero headspacefoil laminate package of this invention. To achieve roughly the samepCO₂ and pO₂ values in the foil laminate packaging process, as occur inthe ampuling process, the foil laminate pouches were filled with QCsolutions that were tonometered under partial vacuum with theappropriate gases, and then the solutions were pumped intozero-headspace foil laminate pouches, and pasteurized as set forthbelow. A limited accelerated stability study was then performed inaccordance with the method described above. The two studies allowed usto make the following comparison:

TABLE 7 Comparison of Packages with and without Headspace Values beloware ΔpO₂, mmHg (except for factors) Condition Level Level Temp. Time, 23 ° C. wks Ampules Bags Factor Ampules Bags Factor 45 2 −1.2 −36 30X−0.9 −12 14X 50 2 −1.0 −42 42X 55 1 −2.6 −53 20X −1.3 −21 16X 60 2 −1.9−57 30X

It can be seen that there is a considerable range among the six factors,from a low of 14× to high three times as great, 42×. What can beconcluded from the data is that maintaining pO₂ stability in arelatively inert, zero headspace package is at least an order ofmagnitude more difficult than maintaining the same degree of pO₂stability in a package with a headspace at least half as large as thesolution volume.

Raw Material Screening Test

A representative screening test for the components of formulations ofthis invention is demonstrated by the study of this section. Tensolutions with the same defined level Of pO₂, were preparedsimultaneously by equilibrating deionized water in glass containers at50° C. in a water bath. The temperature of the water bath must be atleast as high as the temperature intended to be used for the acceleratedtest which is to follow, so as to avoid out gassing of oxygen during thestress cycle at elevated temperature.

In order to magnify the oxygen consumption of individual ingredients,especially in cases where there may be several minor contributors asopposed to one or two major contributors, it is desirable to increasethe concentrations above their normal use levels. In this study, theinventors increased concentrations by five times.

The inventors isolated the eight chemicals added to the two componentformulations in the study described above under the heading Shelf Lifeand Use Life. Those eight chemicals are the inorganic compounds NaCl,KCl, CaCl₂, NaOH, and the organic compounds, citric acid, glucose, MOPS(pH buffer) and ProClin 300 (biocide). However, in order to test in theneutral pH range (6-8) some chemicals had to be tested together, namely,MOPS with NaOH, and citric acid with sodium bicarbonate. For efficiency,the three chloride salts were tested together, based on our predictionthat the inorganic chemicals were unlikely to be significantcontributors to slow oxidation reactions. In addition to the eightchemicals already mentioned, we also tested an alternative pH buffer,HEPES, and two dyes, SRB and Mordant Yellow 7.

Chemicals were added to the pre-warmed deionized water in glass bottles,and mixed by inversion. When all chemicals in all bottles weredissolved, solutions were poured into bags which had been sealed on 3sides, followed immediately by sealing the fourth side below the liquidlevel. After a 44 hr/65° C. pasteurization step, half of the bags wereleft at room temperature while the other half were stressed for twelvedays at 50° C., followed by cooling to room temperature. Controls andstressed bags were tested for pO₂ in one run on two model 288s. Thefollowing results were obtained:

TABLE 8 Screening Test of Chemical Ingredients for Oxygen ReactivitySubstance Mean ΔpO₂ Range Water blank −4 mmHg 4 mmHg MOPS, Sigma −5 4MOPS, Research Organics −4 4 Glucose, Sigma −14 3 Glucose, Fluka −11 3ProClin 300, lot LA60507 −7 2 ProClin 300, lot LA64543 −9 4 Citric Acid,Bicarbonate, Brij −9 10 NaCl, KCl, CaCl₂ −5 4 HEPES (pH buffer) −6 3Sulforhodamine B (red dye) −6 8 Mordant Yellow 7 (dye) −13 5

Those results show that:

1. glucose and Mordant Yellow 7 are the most significant oxygenreactives;

2. ProClin 300 is moderately reactive;

3. MOPS, HEPES, and the three chloride salts are relativelynon-reactive; and

4. results for SRB and the citric/bicarb/Brij mixture werenon-conclusive due to excessive bag-to-bag variability. However, furthersubstantially similar screening showed that SRB was moderately reactive,and that citric acid, sodium bicarbonate and Brij were relativelynon-reactive.

In regard to Mordant Yellow 7, shown above to be significantly oxygenreactive, it can be concluded that it would be preferred that anotheryellow dye or Mordant Yellow 7 that is less oxygen reactive, e.g., fromanother source, be used in the formulations of this invention. When tHbis the only CO-Oximetry analyte to be tested, a red dye is sufficient.

SRB is a red dye, and the particular SRB screened was found to bemoderately reactive. It may be preferred to screen SRBs from othersources or other red dyes for an SRB or other red dye having loweroxygen reactivity. However, the accelerated stability results in Table 4show that the level 3 formulation containing the above-screened SRB andMordant Yellow 7 dyes has significantly more than a year's shelf life.Shelf life of such a formulation may be further prolonged by screeningand incorporating therein dyes having lower oxygen reactivity.

Effect of Glucose on pO₂ Instability

The strong destablizing effect of glucose on pO₂ stability was noted inthe study described below. This study compared two sources of glucose,used at 1.8 g/L—one from Fluka Chemical Corp. [Ronkonkoma, N.Y. (USA)]and one from Sigma Chemical Co. [St. Louis, Mo. (USA)]—in a 150 mmHg pO₂calibrator at pH 6.8 to the same calibrator without any glucose added. Alimited accelerated stability test was conducted on those solutions,with the following outcome.

TABLE 9 Effect on pO₂ of Storing 150 mmHg Calibrator at HighTemperatures for 2 wks Mean difference from Non-heated solutions, mmHgNo Glucose Fluka Glucose Sigma Glucose TEMP added added added 45° C.−2.2 −5.7 −6.3 50° C. −4.7 −8.8 −9.9

It can be seen that:

1. at both temperatures, both sources of glucose at least double the pO₂decrease; and

2. the differences between the two glucose sources are relatively minor.

Thus, those results fit very well with the results reported in thesection on screening raw materials above. Moreover, because the sourceappears to play a relatively minor role, this suggests that the oxygenreactivity is inherent in glucose, which was not obvious before weundertook this study.

There are at least three well-known degradation mechanisms for glucose:

1. reaction with oxygen, forming gluconic acid, if glucose oxidase ispresent;

2. reaction with ATP, forming glucose-6-phosphate, if hexokinase ispresent; and

3. alkaline rearrangement, forming first fructose, later mannose.

The first two are widely used in clinical chemistry assays to measurethe level of glucose in blood. The third, occurring at even mildly basicpH, is the most common route for glucose instability in quality controlsused in conjunction with glucose assays.

None of those three common reactions explain the presumed reactionbetween glucose and oxygen in the formulations of this invention becauseonly one lists oxygen as a reactant, and in that case, the necessaryenzyme is not present in our formulations. Moreover, stoichiometrybetween moles of glucose decrease and moles of oxygen decrease were notnoted, and no 1:1 relationship was found.

TABLE 10 Stoichiometry of Oxygen v. Glucose Decomposition Glucose Oxygen200 Cal mg/dL/wk mmol/L/wk mmHg/wk mmol/L/wk Lot 50° C. −0.78 −.043 −2.7−.004 1645 45° C. −0.41 −.023 −2.0 −.003 Lot 50° C. −0.71 −.039 −3.3−.005 1655 45° C. −0.35 −.019 −1.9 −.003

It can be seen that the glucose loss is considerably greater than theoxygen loss. The additional glucose loss must be due to non-oxygenconsuming reactions.

Film

The film which is used for the container is multilayered and uses amaterial having low or no oxygen reactivity, preferably polypropylene(PP), for the inner layer, aluminum foil for the middle layer, and anouter layer that is protective of the aluminum layer, preferablypolyester. The outer layer merely provides protection for the aluminumlayer, preventing abrasion and corrosion. Thus, for example, a nylonlayer, or even a simple lacquer coating are suitable alternatives.[Nylon is a family of high-strength, resilient synthetic materials, thelong-chain molecule of which contains the recurring amide group CONH.The term “nylon” was coined by its inventors at E. I. duPont de Nemours& Co., Inc.] However, the outer layer should have a melting pointgreater than PP's melting point which is about 170° C.

An important parameter of the aluminum layer is that it be thick enoughso that there are no pinholes, thus preventing physical leakage ofoxygen, yet thin enough so that it can be readily formed into pouches onautomated machines and will, after being filled, release its contentswithout undo force by readily collapsing as the contents are removed.

The inner PP layer is important for several reasons. First, it must meltand form the seal which closes the package. Second, it must beunreactive with the oxygen. It is this second factor which distinguishesthis packaging material from those previously used for this purpose.

To the inventors' knowledge, this laminate has never been usedcommercially for packaging products which contain high-precisionsolutions with dissolved gases for scientific, medical, analyticalpurposes. The PP lined laminate is not known to be used by others as anoxygen barrier for chemical products. A former manufacturer of oxygencalibrators (Mallinckrodt Sensor Systems, Inc., Ann Arbor, Mich.) hasused laminated film to package a calibrator, but they used polyethyleneas the inner, sealing layer. The PP lined laminate has been used in thepast mainly for food products, and has been chosen for the high meltingpoint of the polypropylene sealing layer, which makes this materialsuitable for sterilization in a steam autoclave or similar equipment.

Films from various suppliers were evaluated for efficacy in maintainingthe dissolved gas concentrations of solutions stored within. Films wereobtained from Kapak Corp., Minneapolis, Minn. (part no. 50703), AmericanNational Can Co., Mount Vernon, Ohio (part nos.

M-8309, M-8359, M-8360), James River Corp., Cincinnati, Ohio (part nos.JR 4123, JR 4400), Technipaq, Inc., Crystal Lake, Ill. (“Dull FoilLaminate”), Lawson Mardon Flexible, Inc., Shelbyville, Ky. (spec nos.13362 and 15392), Smurfit Flexible Packaging, Schaumburg, Ill. (LC Flex70459, 70464), and Rollprint Packaging Products, Inc., Addison, Ill.(RPP #26-1045). 4-sided bags were either purchased with 3 sidespre-sealed or were formed using an impulse heat sealer from Toss MachineComponents, Inc., Bethlehem, Pa., Model 01617. The 3-side sealed bagswere filled with various reference solutions and immediately sealedthrough the liquid, allowing no headspace inside the package. In someinstances, for enhanced stability of the oxygen partial pressure in thereference solution stored within the bags, filled, sealed bags wereheat-treated at elevated temperatures between approximately 50° C. and121° C. for times ranging from 15 minutes to 7 days, depending on thetemperature.

FIG. 1 a shows a side view of a sealed bag 1, and one possible locationof the access device 5 in the interior of the bag is shown. The sealedportion of the bag is also shown 6. FIG. 1b shows the 3 layers of apreferred film, the inner polypropylene layer 2, the middle aluminumlayer 3, and the outer polyester layer 4.

Some filled bags were left at room temperature; others were stored atelevated temperatures for various times. To simplify reporting of thisand subsequent trials, we used storage at 55° C. for 1 week as a basisfor comparison. After removing test bags from the incubator, they werecooled to room temperature and tested on two critical care analyzers[generally selected from the 200 Series Critical Care Diagnostic Systemsmanufactured by Chiron Diagnostics Corporation; Medfield, Mass. (USA); a278 was often used with a 288] with control bags in the same run. Inparticular, the pO₂ results were examined in a series of six studies.Due to differences in conditions such as reagent composition and packagesurface-to-volume ratios, the pO₂ differences are not directlycomparable. Therefore, all results were converted to relative scoreswhere the most stable laminate was assigned a score of 1.00, and allother laminates were assigned scores on the basis of ΔpO₂ ratios. Usingthis convention, the following results were obtained:

TABLE 11 Material N Mean Score Range of Scores Polyethylene 4 0.140.10-.016 Polypropylene 6 0.41 0.18-1.00 Polyester 2 0.28 0.26-0.30

The preferred and most preferred laminates have an inner PP liner of thethickness shown below, a middle layer of aluminum as shown below, and anouter polyester layer. (The thickness and material selection of theouter layer is least critical and can vary somewhat.) Acceptable filmthicknesses are also shown. Approximate thicknesses of layers in mils({fraction (1/1000)} inch):

TABLE 12 Polypropylene Aluminum Polyester Most preferred 4 mil 0.5 mil0.5 mil Preferred 2-5 mil 0.5-0.7 mil 0.5 mil Acceptable 1.5-5 mil0.3-1.0 mil 0.1-2 mil

Other acceptable layers include polyester at 0.5-2 mil for the innerlayer; for the outer layer either nylon with thickness of 0.2-2 mil orlacquer coating. Polyethylene has not been found to be acceptable as aninner layer.

There are detrimental properties that result if any of the film layersare too thick. Namely, the laminate becomes too rigid, making itdifficult to form and fill during manufacture, and difficult to pump outthe liquid contents from the pouch/bag during use. Furthermore, if thealuminum layer is too thin, there is a higher probability of havingpin-holes, which may lead to gas leakage. If the sealing layer is toothin, it may be entirely displaced at the moment of heat-sealing at theseal under high pressure required for strong seals, thereby exposingbare aluminum which would react with oxygen.

Stability testing has shown that the PP lined film is preferred over thepolyethylene film. The Arrhenius method of predicting product shelflifeis well-established in the in-vitro diagnostics and pharmaceuticalindustries (Conners et al, “Chemical Stability of Pharmaceuticals: AHandbook for Pharmacists”, NY: Wiley, 1986; Porterfield & Capone, MD&DI45-50, April 1984; Anderson & Scott, Clin. Chem. 37: 3, 398-402, 1991;Kirkwood, Biometrics. 33, 736-742, December 1977). Products are storedat elevated temperatures for various times, following which they arere-equilibrated at ambient temperature and tested against non-stressedcontrols for critical properties such as activity of a component ormeasured analyte. The rate of change or more conveniently, thetime-to-failure, of a given analyte is determined for each temperature,often by plotting log(C/Co) vs. time, which is a linear function for themost common, first-order reactions. Owing to the linear relationshipbetween log(time-to-failure) and the inverse of the absolute temperature(1/K), a plot can be constructed from the elevated-temperature data, andthe resulting line can be extended to the maximum recommended storagetemperature to predict the time-to-failure at that temperature. In thismanner, actual shelflife can be predicted in advance.

In an early predicted shelflife study using polyethylene-lined bags,finished packages filled with an oxygen reference solution were storedat 35, 45, and 55° C. for times ranging from 4 days to 8 weeks,depending on the storage temperature, using longer times with lowerstorage temperatures. Each test condition included 4 bags tested on twoblood gas analyzers [200-series manufactured by Chiron Diagnostics Corp.(CDC), supra]. Time-to-failure (TTF) was defined as a 2% change in pO₂.

TABLE 13 Polyethylene (PE) Temperature 1/K Time-to-Failure Log (ttf) 55°C. .0030488 0.6 weeks −0.222 45° C. .0031447 1.1 weeks 0.036 35° C..0032468 4.4 weeks 0.647

Regression analysis on the above data, based on plotting log(ttf) as afunction of 1/K, results in a predicted 25° C. shelflife of 3 months foran oxygen reference solution stored in the polyethylene-lined bag. Thecorrelation coefficient, r, is 0.98.

In the polypropylene study, finished packages containing an oxygenreference solution were stored at 35, 40, 45, and 50° C. for timesranging from 1 to 9 weeks, depending on the storage temperature, usinglonger times with lower temperatures. Each test condition included 3bags tested in singlicate on two blood gas analyzers (200-series fromCDC, supra). The first-order model was used to determine time-to-failure(TTFs), where failure was defined as a 2% change in pO₂.

TABLE 14 Polypropylene (PP) Temperature 1/K Time-to-Failure Log (ttf)55° C. .0030960 1.3 weeks 0.106 45° C. .0031447 3.3 weeks 0.521 40° C..0031949 5.7 weeks 0.755 35° C. .0032468 12.3 weeks  1.091

Using the four TTFs, an Arrhenius plot was constructed (see FIG. 5),where time to failure (in weeks) (TTF) is shown as a function of inversetemperature, 1/K (shown as T in FIG. 5). (1/K is the inverse of Kelvintemperature.) The linear extrapolation to 25° C. is 61 weeks or 14months, for an average pO₂ change of −0.066 mmHg/wk. The reliability ofthe prediction is affirmed by the highly linear relationship among the 4points, with a correlation coefficient, r, of 0.99. A score of 1.00would indicate that all points fall on a straight line; a score of 0.00,that no relationship exists between log ttf and 1/K. (Note that theequation for the Arrhenius plot exemplified was found to be logy=−19.48+6339x.)

The resulting predicted shelflife of the oxygen reference solution inpolypropylene-lined bags represents a four-to-fivefold improvement overthe shelflife predicted for oxygen reference solution stored in thepolyethylene-lined bags. It also represents a nearly tenfold improvementover a recent state of the art product, known as “Cal B” which was soldby Mallinckrodt Sensor Systems, Inc. [Ann Arbor, Mich. (USA)]. Thesoftware in the GEM® Premier Analyzer that accompanies that systemautomatically subtracts 0.58 mmHg pO₂ from the initial assigned pO₂ forevery week which has elapsed since manufacturing in order for the Cal Bcalibrator to be useable for its expected commercial usage period. Ifnot for this calculation, using our 2% criterion, the useful shelflifewould be only 7 weeks, clearly too short a time for commercial use ofthe product. Moreover, note that the actual Cal B shelflife, 6 months,limits the shelflife of the entire cartridge to only six months,arguably the minimum practical shelflife for an in-vitro diagnosticproduct. On the other hand, 14 months is clearly an acceptableshelflife.

Other factors which discourage use of PP-lined laminates are theirgreater stiffness and higher melting points. PP durometer hardness, onthe Shore D scale (ASTM Designation: D 2240-91 American Society forTesting and Materials, Philadelphia, Pa.), is 70-80 compared with only44-48 for PE. Stiffness impedes high surface:volume ratio, whichimproves shelflife, and makes automation on form/fill/seal machines moredifficult. The higher melting point for PP, 171° C. compared to only137.5° C. for PE, requires more energy, time, or both to seal the bags.

Other variations in the packaging method are possible. For example,other shapes of packages that reduce the ratio of surface area ofpackage to volume of solution and gas within the package (e.g., 2circular pieces of film which are sealed together), would reduce evenfurther the exposure of the solution and gas to the film, even furtherreducing the oxygen degradation. The packaging disclosed herein is alsoeffective in protecting tonometered solutions containing other gasesaside from oxygen. Furthermore, various configurations of package (e.g.,three-sided seal or side-seam; four-sided sealed; gusseted packages; or“stand-up, pouches) can be used. (Compare, for example, FIG. 1c, whichshows 4 sides sealed, to FIG. 1d, which shows a 3-sided seal.) Thesepackage variations affect utility of the packaging method and are notsimply design alternatives. Other variations will be apparent to thosewith expertise in this technology area.

The Access Device

The access device is attached inside of the package. Attachment can beachieved using any technique available, for example, via use ofadhesive, heat-bonding, ultrasonic welding, etc. This access device isan optional component of the package and is particularly useful when thecontents of the container are used over a period of time after aprolonged storage interval. In previous approaches, a valve has beensealed into the edge or through the wall of the container so that itwould be accessible from the outside of the container. However, in thepackage used herein, the access device is sealed totally within thepackage on the inner wall, and does not breach the seal or the walls ofthe container.

FIGS. 1a, 1 c and 1 d show typical locations for the access device. FIG.2 shows the detail of a typical access device, with 7 being the portionof the access device sealed to the wall of the container, 8 being theouter portion of the delivery channel, 9 being the inner portion of thedelivery channel, and 10 being the sealed portion of the deliverychannel which is punctured by the probe, which then makes a tight fitwith the inner portion of the delivery channel, thus preventing leakagefrom the container. FIG. 3 shows a typical probe, which is used topuncture the bag and fit into the access device inside the bag, with 11representing the probe and 12 representing the sharp end of the probewhich punctures the sealed portion of the delivery channel. The probe isincorporated in a clamping device 13 (see FIGS. 4a, 4 b and 4 c) whichhas a circular opening 14 which fits over the hemispherical back of theaccess device 15 aligning the probe with the delivery channel. The probeis connected to other components which allow the oxygen referencesolution to flow to the apparatus where it can be utilized in assays.When the package is punctured, the probe pierces the wall and forms atight seal with the delivery channel of the access device. Before thepackage is punctured, the access device is totally isolated within the(more or less) impermeable walls of the container. This approach has anadvantage over other valves and access devices in that it does notprovide a diffusion pathway to the outside environment. Obviously therecan be variations in the design of the access device and probe, whichwill be apparent to those with skill in the art.

The access device is also made of PP so that it seals well with the wallof the container. The description of the access device should allow forsome variations of the preferred access device. For example, the accessdevice might be sealed to both walls of the package to provide an addedbenefit of stabilizing the shape or the package. The access device canbe sealed at any location inside the container, for example, in a corner(for ease of attaching a clamp) or away from the edge of the container.Furthermore, the access device does not need to be attached to thecontainer if there is some technique incorporated for locating theaccess device. For example, if the access device were to contain anembedded magnet, the application of an exterior magnet could be used tocapture and position the access device. Other shapes (cones, indents,etc.) might be used for the locating feature. Rings can be molded intothe inner wall of the delivery channel to improve the seal afterpuncture. The travel distance of the probe can be limited to preventpuncture of the adjacent wall of the container.

Tubing

The access device of the packaging of this invention extends the uselife of oxygen reference solutions. Once the packaging is opened, theaccess device is designed to minimize oxygen diffusion therebyincreasing the use life of the reference solution. Further, flexible andrelatively gas impervious tubing is used to minimize oxygen diffusion.

The tubing conveys the oxygen reference solution from the packagethrough the pierce probe (FIG. 3) to the analyzer. For example, in FIG.3, such tubing would have a diameter which fits tightly into the secondof the three cylindrical regions, wherein the third cylindrical regionhas the same diameter as the internal diameter of the tubing(illustrated with broken lines in FIG. 3) that intersect the pierceprobe (11).

It is preferred that the durometer (Shore D scale) of such tubing be ina range of from 10 to 100, preferably from 70 to 94, and more preferablyfrom 80 to 84. Condensation polymers having the requisite durometercharacteristics are preferred, particularly preferred are polyamidecondensation polymers, more preferred are polyester/polyether blockco-polymers or polyester elastomers. Especially preferred tubing isNylon™ [DuPont; Wilmington, Del. (USA)] and Hytrel™ 8238 [DuPont].

Representative experiments below are described wherein tubing materialscan be tested for suitability for use in the methods of this invention.Silicone, fluoropolymers and plasticized polyvinylchloride were therebydetermined not to be suitable tubing materials.

Use Life—Selecting Tubing Material

Similar to shelf life, which is often limited by pO₂ due to reaction ofoxygen with packaging or contents, use life is also often limited bypO₂, but by a different mechanism—diffusion. The effectiveness of theaccess device design of the foil laminate packaging of this inventionminimizes pO₂ diffusion. This study employed two flexible tubingmaterials—Hytrel 6356 [DuPont] and Zytel 42 Nylon [DuPont]. That tubingwas used to conduct the oxygen reference solution from the probe of FIG.3 (as discussed above) which fits into the access device of the foillaminate pouch to the analyzer (M288 model from CDC, supra).

An open bag use life test was run on the following formulation which hada pO₂ of 40 mmHg:

NaHCO₃ 20 mmol/L NaCl 65 mmol/L KCL 3.2 mmol/L CaCl₂ 2.8 mmol/L CitricAcid 1.7 mmol/L LiCl 6 mmol/L MOPS 40 mmol/L Brij 700 0.05 g/L CosmocilCQ 0.10 g/L.

The pO₂ equilibrium point is approximately 190 mmHg at 22° C. whenmeasured at 37° C. The lower pO₂ within the bag increases the drivingforce for oxygen from room air to diffuse into the bag and thereby intothe test solution. Six bags were tested over a 28-day period using 2M288s [CDC, supra]. Results are summarized in the table below and inFIG. 6.

TABLE 15 Use Life of Very Low PO₂ Solutions in Bags with Nylon v. HytrelTubing Change to pO₂ over 28 days Hytrel 6356 −0.9 mmHg Bag 1 +3.5 mmHg−0.9 mmHg Bag 2 +0.7 mmHg −1.3 mmHg Bag 3 +3.0 mmHg +2.1 mmHg Mean +2.4mmHg ±0.0 mmHg

A reasonable tolerance limit for allowable pO₂ change is ±4 mmHg at thislow pO₂. It can be seen that all six bags performed within this range,but the bags with Nylon tubing attached had, on average, less increasein pO₂ over the test period.

The inventors' best explanation for the greater stability of pO₂ in thebags with Nylon tubing attached is that the Nylon has a higherdurometer, or hardness, than the Hytrel 6356. Using the Shore D scale(ASTM Designation, supra), Zytel 42 Nylon (Dupont) is rated 82 comparedwith 63 for Hytrel 6356. The higher durometer implies that the moleculesof the nylon are packed more tightly together both making the materialmore rigid and making it more difficult for gas molecules to diffusethrough the interstitial spaces. Therefore, Zytel 42 Nylon, andpresumably other nylons are preferred tubing materials. Also, Hytrel8238 has the requisite durometer and is a preferred tubing material.

Further experiments with tubing materials were performed, whereinaqueous solutions were tonometered with a gas mixture containing nooxygen, aspirated into a section of test tubing sufficient to contain100 uL using a syringe, held in the tubing for 60 seconds, and thenaspirated into a model 288 analyzer [CDC, supra] beyond the segmentationvalve by manually turning the pump roller. The resulting pO₂ readingsserved as indicators of the degree to which oxygen from the tubingdiffused into the aqueous solutions. More than 15 tubing materials weretested in this manner. The results indicated that polyester/polyetherblock co-polymers, notably Zytel 42 Nylon and Hytrel 8238, are preferredtubing materials. Another preferred tubing material is Saran™[polyvinylidene chloride; Dow Chemical Company; Midland, Mich. (USA)].Silicone, fluoropolymers and plasticized polyvinylchloride were foundnot to be suitable as tubing materials.

Reactivity of Oxygen with Polypropylene

Oxygen is much less reactive with PP than it is with polyethylene. It isthis lower reactivity that makes PP a more desirable material to be usedas an inner layer of the foil laminate packaging of this invention. Inthe past, developers were concerned with permeability of the inner layerto oxygen, but this turns out, however, to be a less important attributethan the reactivity for this type of reference solution.

Both PP and PE provide reasonable sealing, although the PP has a highermelting temperature. In addition, both materials provide equivalentprotection against liquid leakage. However, in polyethylene, there ismore reactivity between oxygen and the polymer, thus reducing the oxygenlevel. It is not permeation through the polyethylene film that waslargely responsible for reducing the oxygen level. This argument isbased on the following numbered points.

1. Although the pO₂ level in the oxygen reference solution seems to beconsiderable, at roughly 200 mmHg, in molar terms, it is only 0.27mmol/L. The calculation to convert from mmHg partial pressure to mmol/Loxygen concentration is reasonably simple and straightforward, butoxygen is rarely described in the literature in molar units. Rather,where it is not in partial pressure units such as mmHg or kPa, it isfound in concentration units such as mg/L or mL/dL. However, approachingthe oxygen loss problem from the molar perspective teaches us thatreaction of only 0.005 mmol/L (2%) would cause product failure.Ultraviolet (UV) spectroscopy studies showed that at elevatedtemperatures, water-soluble, UV-absorbing substances are extracted fromthe sealing layer into the bag contents. This is true for both PP- andPE-lined bags. Finally, whereas only 0.005 mmol/L reactant is requiredfor product failure (by pO₂ decrease), with 100 mL reagent in a 4″×6″bag, only 0.1% of an additive with a molecular weight of 500 in a 4 milPP film would provide 0.05 mmol/L of oxidizable reactant, ten times theamount needed to explain a 2% decline in pO₂. Thus, the stoichiometry isreasonable, even assuming an extraction efficiency of only 10%.

2. PP sealing layers from different vendors differ markedly in the pO₂changes in oxygen calibrator sealed within them when they are subjectedto elevated temperatures, as demonstrated in Table 11 above. Yet thepermeability of polypropylene roll stock from any of the several vendorscan be expected to be similar because it should be a property of thebulk polymer, unless it has been modified into an orientedpolypropylene. (Oriented PP is not known to be laminated to aluminumfoil.) Thus, it is unlikely that permeability differences can explainthe differences in pO₂ deltas shown in Table 11. However, since thevarious PP vendors are known to use a considerable variety of additivesto the basic PP resin (these additives being nearly always proprietary),it is quite likely that differences in additives among the variousresins explain a considerable portion of the differences in pO₂ deltas,as different additives or even the same additives in differentconcentrations would react to a greater or lesser degree with the oxygenin the calibrator.

3. The most convincing evidence to support the importance of reactivityover permeability is from an experiment which isolated the two effects.A uniform population of 3-side-sealed PP-lined bags were filled with anoxygen calibration solution tonometered such that oxygen partialpressure would be roughly 200 mmHg. A control group of the same bags wasfilled normally and immediately sealed on the Toss impulse sealer. Twotest groups had five pieces, cut so as to just fit into the bag, ofeither polyethylene or polypropylene added to the bags just beforefilling and sealing. As in the stability tests described above, somebags from all three groups were left at room temperature, while others,randomly selected, were stored at 55° C. for 1, 2, and 3 weeks. Bagswere cooled to and allowed to equilibrate at room temperature for atleast 24 hours, and then tested in the usual manner, that is, intriplicate on two 200-series blood gas analyzers [CDC, Medfield, Mass.(USA)], alternating during runs between control and test conditions. Thefollowing results were obtained:

Stress pO₂, Mean Net Test Group Condition (SD) ΔpO₂ ΔpO₂ Control Control201 (3) mmHg −10 mmHg 3 wks at 191 (1) mmHg 55° C. +PolypropyleneControl 219 (3) mmHg −13 mmHg  −3 mmHg 3 wks at 206 (6) mmHg 55° C.+Polyethylene Control 221 (2) mmHg −42 mmHg −32 mmHg 3 wks at 179 (6)mmHg 55° C.

The effect of the polyethylene on pO₂ is both dramatic, being an orderof magnitude more severe than polypropylene, and significant, with theadditional 29 mmHg decrease being nearly five times the greatest SD, 6mmHg. Permeability cannot explain this difference because the plasticsheets were contained entirely within the bags.

The description of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to explain the principles of the invention and itspractical application to enable thereby others skilled in the art toutilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto.

All references cited herein are hereby incorporated by reference.

What is claimed is:
 1. A zero headspace container that maintains astable partial pressure of oxygen in a multi-analyte reference solutionhoused therein, comprising: (a) an inner layer forming the interior ofthe container and composed of a polymeric material that does not reactwith oxygen or has an oxygen reactivity that is sufficiently low so asto maintain the partial pressure of oxygen of a multi-analyte referencesolution therein at a specified value ±4 mm Hg; (b) an intermediatelayer comprised of aluminum; (c) an outer layer that serves to protectthe intermediate layer from physical damage; (d) a multi-analytereference solution housed within the interior of the container; and (e)an access device housed totally within the interior of the container. 2.The container of claim 1, wherein the inner layer is polypropylene thatexhibits low or no oxygen reactivity.
 3. The container of claim 2,wherein the partial pressure of oxygen is maintained at the specifiedvalue ±4 mmHg at room temperature for at least six months.
 4. Thecontainer of claim 2, wherein the inner layer has a thickness ofapproximately 1.5 mil to 5 mil, the intermediate layer has a thicknessof approximately 0.3 mil to 1 mil and the outer layer has a thickness ofapproximately 0.1 to 2 mil.
 5. The container of claim 4, wherein theinner layer has a thickness of about 2 to 5 mil and the intermediatelayer has a thickness of about 0.5 to 0.7 mil.
 6. The container of claim5, wherein the inner layer has a thickness of about 4 mil, theintermediate layer has a thickness of about 0.5 mil and the outer layeris either a lacquer coating, polyester, or nylon.
 7. The container ofclaim 2, wherein the outer layer is polyester.
 8. The container of claim1, wherein the specified value is between 20 mmHg and 700 mmHg and thepartial pressure is maintained at the specified value ±4 mmHg at roomtemperature for at least six months.
 9. The container of claim 1,wherein the partial pressure of oxygen is between 20 mxnHg and 700 mmHg,inclusively.
 10. The container of claim 1, wherein the partial pressureof oxygen is between 30 mmHg and 500 MmHg, inclusively.
 11. Thecontainer of claim 1, wherein the access device is connected to one ormore walls of the container.
 12. The container of claim 1, wherein thecontainer is in the form of a 3-side, center-sealed pouch.
 13. Thecontainer of claim 1, wherein the multi-analyte reference solutioncalibrates or controls for partial pressure of oxygen and one or moreother analytes selected from the group consisting of pH, pCO₂,electrolytes, metabolites, tHb, CO-Ox fractions and hematocrit.
 14. Thecontainer of claim 13, wherein the metabolites are selected from thegroup consisting of glucose, lactate, bilirubin, urea and creatinine.15. The container of claim 1, wherein the multi-analyte referencesolution consists essentially of chemicals having low or no oxygenreactivity.
 16. The container of claim 15, wherein at least some of thechemicals are organic.
 17. The container of claim 16, wherein thepartial pressure of oxygen is between 20 mmHg and 700 mmHg, inclusively.18. The container of claim 17, wherein the partial pressure of oxygen isbetween 30 mmHg and 500 mmHg, inclusively.
 19. A zero headspacecontainer that maintains a stable partial pressure of oxygen in amulti-analyte reference solution housed therein, comprising: (a) aninner layer forming the interior of the container and comprised of apolymer; (b) an intermediate layer comprised of aluminum; (c) an outerlayer that serves to protect the intermediate layer from physicaldamage; (d) a multi-analyte reference solution housed within theinterior of the container; and an access device housed totally withinthe interior of the container, wherein the inner layer and themulti-analyte reference solution are free from any component having anoxygen reactivity that is sufficiently destabilizing so as to allow thepartial pressure of oxygen of the multi-analyte reference solution todeviate from a specified value by at least 4 mm Hg.
 20. A method formaintaining a stable partial pressure of oxygen in a multi-analytereference solution, the method comprising: (a) introducing the referencesolution into a laminated container comprising (i) an inner layer thatserves as the interior of the container and is composed of a polymericmaterial that does not react with oxygen or has an oxygen reactivitythat is sufficiently low so as to maintain the partial pressure ofoxygen of the reference solution at a specified value ±4 mm Hg, (ii) anintermediate layer of aluminum, (iii) an outer layer that serves toprotect the intermediate layer from physical damage, and (iv) an accessdevice housed totally within the interior of the container; and (b)sealing the container so as to provide zero headspace therein.
 21. Themethod of claim 20, wherein step (b) comprises forming the containerinto a 3-side, center-sealed pouch.
 22. The method of claim 20, whereinstep (b) comprises heating and fusing the inner layer so as to seal thecontainer.
 23. The method of claim 20, wherein the access device enablesremoval of the solution from the container's interior.
 24. The method ofclaim 23, further comprising, after step (b), piercing the access devicewith a probe that is connected to an analyzer through tubing that has adurometer in the range of 10 to
 100. 25. The method of claim 24, whereinthe tubing has a durometer in the range of 70 to
 94. 26. The method ofclaim 25, wherein the tubing has a durometer in the range of 80 to 84.27. The method of claim 24, wherein the tubing is a polyamidecondensation polymer.
 28. The method of claim 24, wherein the tubing isa polyester/polyether block co-polymer or a polyester elastomer.
 29. Themethod of claim 20, wherein the access device is composed of a polymericmaterial that does not react with oxygen or has an oxygen reactivitythat is sufficiently low so as to maintain the partial pressure ofoxygen of the solution at a specified value ±4 mm Hg.
 30. A method formaintaining a stable partial pressure of oxygen in a multi-analytereference solution, the method comprising: (a) introducing the referencesolution into a laminated container comprising (i) an inner layer thatserves as the interior of the container and is comprised of a polymer,(ii) an intermediate layer of aluminum, (iii) an outer layer that servesto protect the intermediate layer from physical damage, and (iv) anaccess device housed totally within the interior of the container,wherein the inner layer and the reference solution are free from anycomponent having an oxygen reactivity that is sufficiently high so as toallow the partial pressure of oxygen of the multi-analyte referencesolution to deviate from a specified value by at least 4 mm Hg; and (b)sealing the container so as to provide zero headspace therein.